Voltage-activated potassium (Kv) channels are two-domain proteins comprised of the voltage-sensing domain (VSD) and the pore domain (PD). These domains appear to be structurally independent except for electromechanical coupling that transfers VSD conformational changes into PD gating. Recent crystallographic structures show the VSDs in the open or so-called up-state. Two crucial questionswhich underlie this Program Projectare (a) the structure and orientation of the Kv VSDs in the closed (downstate) and (b) how the VSDs move between the two states. Recent evidence strongly suggests that the phosphate groups of the membrane phospholipids are crucial for state switching due to interactions between lipid phosphate groups and the arginines on the VSD S4 helix. Lipid-protein interactions thus appear fundamental to channel gating. Indirect information about these lipid-protein interactions can be obtained using tarantula toxins, such as VSTxl, that inhibit the activation of Kv channels by binding to the VSD paddle motif mediated by simple water-to-membrane partitioning, suggesting that the toxins bind to the paddle at the protein-lipid interface. But little is known about their molecular interactions with membranes, such as how they are positioned within membranes, what effect they have on membrane structure, or how they dock to VSDs within the membrane. This Project addresses basic aspects of the molecular interactions of VSDs and tarantula toxins with lipid bilayers using neutron diffraction in concert with the molecular dynamics (MD) simulations of Project 1. It also lays a foundation for neutron and x-ray studies of VSD conformational changes in supported bilayers under the influence of electrochemical gradients (Project 3). The Specific Aims of this Project are as follows: (1) Determine the disposition of the KvAP VSD and related proteins in oriented lipid bilayers using neutron diffraction and specific deuteration. The experiments will ultimately provide critical information about the position and orientation of the S3b-S4 voltage-sensing paddle. (2) Determine the interface connections and bilayer perturbations of synthetic VSD S4 helix and related helices incorporated into oriented lipid bilayers using specific deuteration and neutron diffraction. The experiments will clarify the role of lipid phosphates in Kv channel gating. (3) Determine the orientation and penetration depth of the tarantula toxin VSTxl in oriented lipid bilayers using specific deuteration and neutron diffraction. These experiments will reveal the orientation and penetration depth of VSTxl in lipid bilayers and the bilayer perturbations caused by VSTxl. which will provide insights into VSTxl-VSD interactions.